Betulinic acid (BetA) and betulin come from a variety of botanical sources such as bark from Betula alba, Platanus orientalis, Corylus avellana, Carpinus betulus, Alnus glutinosa as well as from Ziziphus sp. Rhamnaceae (Jiri Patocka, Journal of Applied Biomedicine, 2003, 1, 7-12. E. L. Ménard et al., Helvetica Chimica Acta, 1963, XLVI, 1801-1811).
BetA is thus found in many plant species, although in a low concentration compared to betulin. The rare exception is the rich content of BetA in a clover species named Menyanthes trifoliate, which is a bog plant (C. Huang et al., Yao Xue Xue Bao, 1995, 30, 621-626).
Methods of synthesis of many derivatives of BetA and betulonic acid have been described, including amino acid and amide derivatives (Darrick S. H. L. Kim et al., Bioorganic & Medicinal Chemistry Letters, 1998, 8, 1707-1712. O. B. Flekhter et al., in Bioorg Khim., 2004, 30, 89-98; and in Russian Journal of Bioorganic Chemistry, 2004, 30, 80-88), as well as hydrophobic derivatives.

A cyclopropane derivative of betulin has been converted by oxidation into the corresponding BetA and betulonic acid derivatives. The 20,29-dihydro-20,29-dichloromethylene betulinic acid proved to be the most cytotoxic toward human melanoma and human ovarian carcinoma cell lines (A. V. Symon et al. in Bioorg. Khim., 2005, 31, 320-325; and in Russian Journal of Bioorganic Chemistry, 2005, 31, 286-291).
The potential value of triterpenoids, especially pentacyclic triterpenes like betulin and BetA, in the induction of apoptosis in malignant tumour cells has been recognised for some years now (Emily Pisha et al., Nature Medicine, 1995, 1, 1046-1451. Simone Fulda et al., in Cancer Res., 1997, 57, 4956-4964; in J. Biol. Chem., 1998, 273, 33942-33948; and in Int. J. Cancer, 1999, 82, 435-441).
BetA demonstrates selective cytotoxicity against melanoma cells and other malignant cells of neuroectodermal origin (Tino Galgon et al., Exp. Dermatol., 2005, 14, 736-743). Growth inhibition is evident in all neoplastic cell lines and is independent of the status of the apoptotic inducer protein, p53 (Valentina Zuco et al., Cancer Lett., 2002, 175, 17-25).
Tumoural tissue grows when the equilibrium between cell replication and cell death (apoptosis) is not maintained. The immunological mechanisms that control such cellular cycles are complex, being based on the activity of a variety of cytokines as well as on the expression of certain genes.
Two genes are of primary importance:                1. The gene p53, called tumour-suppressor, which codes for the apoptotic inducer protein, p53; and        2. The gene Bcl-2, a proto-oncogene encoding the Bcl-2 family of proteins. Bcl-2 proteins inhibit apoptosis by inhibiting caspase activities. Failure in cancer therapy has been linked to high expression of the Bcl-2 gene (D. Maslinska, Neurol. Neurochir. Pol., 2003, 37, 315-326; A. Linjawi et al., J. Am. Coll. Surg, 2004, 198, 83-90; J. Huang et al., Biol. Pharm. Bull., 2005, 28, 2068-2074).        
In addition to this genetic regulation, different membrane proteins play a part in the control of the cell cycle by acting as receptors for cytokines that regulate apoptosis. Among these cytokines are tumour necrosis factor (TNF) and nerve growth factor (NGF). The binding of such cytokines to their specific receptors induces the activation of caspases, which in turn leads to the proteolysis of a variety of substrates including the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Hydrolysis of PARP induces apoptosis. It is important to note that this mechanism by-passes the proto-oncogene, Bcl-2 (the protein products of which inhibit caspase activities).
The mitochondrion also plays an important role in apoptosis. In 1998, S. Fulda et al. described BetA as a cytotoxic agent that triggers apoptosis by a direct effect at the mitochondrial membrane level, even when the caspases are chemically inhibited (J. Biol. Chem., 273, 33942-33948). BetA directly induced loss of mitochondrial transmembrane potential; soluble cytochrome c excreted in the cytoplasm of the cell thus activated caspases 9 and 3 leading to apoptosis. Action of BetA is thus Bcl-2 independent (V. Zuco et al., Cancer Lett., 2002, 175, 17-25).
In 1999, S. M. Swanson et al. suggested that metabolism of BetA is not necessary for the induction of apoptosis in melanoma cells and that metabolites of BetA are not responsible for its specificity in inducing apoptosis in cancer cells (S. M. Swanson et al., Proc. Amer. Assoc. Cancer Res., March 1999, 40).
Recently, BetA was recognised as a selective inhibitor of human melanoma growth and was reported to induce apoptosis of these cells (Darrick S. H. L. Kim et al., Bioorganic & Medicinal Chemistry Letters, 1998, 8, 1707-1712; E. Pisha et al., Nature Medicine, 1995, 1, 1046-1051; S. Fulda et al., Cancer Res., 1997, 57, 4956-4964). The growth inhibitory action of BetA was more effective against melanoma cell lines than against normal melanocytes. This was recently confirmed in mice bearing human melanoma xenografts (D. A. Eiznhamer and Z. Q. Xu, Drugs, 2004, 7, 359-373).
The anti-proliferative action of BetA seems to be independent of the p53 status and, despite the induction of apoptosis, the expression of the anti-apoptotic protein Mcl-1 is induced (Edgar Selzer et al., Journal of Investigative Dermatology, 2000, 114, 935-940).
Furthermore, a recent publication indicates that BetA activates the transcription factor NF-kappaB in a variety of tumour cell lines and induces apoptosis in a cell-type dependent manner (Hubert Kasperczyk et al., Oncogene, 2005, 24, 6945-6956).
Another study showed that BetA suppresses NF-kappaB activation as well as NF-kappaB regulated gene expression induced by carcinogens, TNF, interleukin-1 (IL-1) and oxidative stress (Yasunari Takada, Bharat B. Aggarwal, J. Immunol., 2003, 171, 3278-3286).
The inhibition of HIV-1 replication by BetA and some other triterpenoids has also been described (Erik De Clercq, Rev. Med. Virol., 2000, 10, 255-277. Chaomei Ma et al., Chem. Pharm. Bull. (Tokyo), 1999, 47, 141-145. Taisei Kanamoto et al., Antimicrob. Agents Chemother., 2001, 45, 1225-1230).
A new derivative, 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB), blocks HIV-1 maturation by inhibiting the cleavage of the capsid precursor, CA-SP1, which leads to a defect in viral core condensation of the viral particles (Donglei Yu et al., Expert Opin. Investig. Drugs, 2005, 14, 681-693).
Recently, Boc-lysinated betulonic acid has been found to be useful in the treatment of cancer, in particular prostate cancer (Brij B. Saxena, Bioorganic & Medicinal Chemistry Letters, 2006, 14, 6349-6358).
A recent review article by R. Mukherjee et al. (Anti-Cancer Agents in Medicinal Chemistry, 2006, 6, 271-279) studies the structure activity relationship of a number of betulinic acid derivatives. The paper concludes that the C-28 carboxylic acid functionality is essential for eliciting cytotoxicity, that a C-3 ester functionality enhances cytotoxicity, and that a C-2 halo-substituent improves cytotoxicity.
Nevertheless, there is of course always a need for alternative compounds for the treatment of diseases such as cancer and viral infections.
It has now been found that betulonic acid derivatives, in particular betulonic acid esters, dihydro-betulonic acid esters, PAG-modified betulinic acid derivatives, and PAG-modified dihydro-betulinic acid derivatives, are useful in the treatment of cancer and viral infections such as HIV, HSV and influenza infection. The ketone functionality of the betulonic acid esters and the dihydro-betulonic acid esters allows these esters to be derivatised and bound to a poly(alkylene glycol), for example, poly(ethylene glycol) or monomethoxy poly(ethylene glycol), which provides the esters with improved solubility and stability in vivo.